Construction of a bacterial artificial chromosome library containing large EcoRI and HindIII genomic fragments of lettuce
Construction of a bacterial artificial chromosome library containing large EcoRI and HindIII genomic fragments of lettuce
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DOI：
10.1007/s001220050428
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发表时间：
1997-03-01
影响因子：
5.4
通讯作者：
Michelmore, RW
中科院分区：
文献类型：
--
作者：
Frijters, ACJ;Zhang, Z;Michelmore, RW
现有的细菌人工染色体（BAG）载体进行了修改，具有独特的EcoRI克隆位点。这为从用多种酶消化的基因组DNA产生代表性文库提供了额外的位点。用HindIII或EcoRI部分消化莴苣基因组DNA，构建莴苣BAC文库。对实验参数进行了优化。由6个连接构建了超过50，000个克隆的BAC文库，代表一到两个基因组当量;每个连接的平均插入片段大小在92.5和142 kb之间变化，组合的平均插入片段大小为111 kb。该文库筛选与抗病基因的标记，这确定了134个BAC克隆从四个地区含有抗性基因。与低拷贝基因组序列连接的抗性基因杂交检测到的克隆比预期的基因组大小的估计。叶绿体和线粒体序列杂交的缺乏表明，该文库主要由核DNA组成。BAC载体中独特的EcoRI位点应允许BAC克隆与利用EcoRI消化的其它技术（例如AFLP（TM）标记和RecA辅助的限制性内切酶（RARE）切割）整合，以从基因组DNA克隆特异性大EcoRI片段。
Existing bacterial artificial chromosome (BAG) vectors were modified to have unique EcoRI cloning sites. This provided an additional site for generating representative libraries from genomic DNA digested with a variety of enzymes. A BAC library of lettuce was constructed following the partial digestion of genomic DNA with HindIII or EcoRI. Several experimental parameters were investigated and optimized. The BAC library of over 50,000 clones, representing one to two genome equivalents, was constructed from six ligations; average insert sizes for each ligation varied between 92.5 and 142 kb with a combined average insert size of 111 kb. The library was screened with markers linked to disease resistance genes; this identified 134 BAC clones from four regions containing resistance genes. Hybridization with low-copy genomic sequences linked to resistance genes detected fewer clones than expected from previous estimates of genome size. The lack of hybridization to chloroplast and mitochondrial sequences demonstrated that the library was predominantly composed of nuclear DNA. The unique EcoRI site in the BAC vector should allow the integration of BAC cloning with other technologies that utilize EcoRI digestion, such as AFLP(TM) markers and RecA-assisted restriction endonuclease (RARE) cleavage, to clone specific large EcoRI fragments from genomic DNA.